A contrast agent for computed tomography and process of preparation thereof

Abstract

The present invention relates to a contrast agent for computed tomography and process of preparation thereof. The composition comprises 30 - 40% of 2, 3, 5-triiodobenzoic acid, 20 - 25% of α-tocopherol, 10 - 20 % coconut oil, and 3 -10% polyethylene glycol. The method of preparation comprises steps of, preparation of α-tocopheryl 2,3,5-triiodobenzoate, preparation of an emulsion, incorporation of α-tocopheryl 2,3,5-triiodobenzoate with emulsion. The contrast agent of the present invention show outstanding contrasting properties, biocompatibility and low toxicity, long residence time and high opacification.

Description

A CONTRAST AGENT FOR COMPUTED TOMOGRAPHY AND PROCESS OF PREPARATION THEREOF FIELD OF INVENTION

[0001] The present invention relates to contrast agents for medical diagnostics. More specifically the invention relates to emulsion-based contrast agent which is of nano scale. The invention also relates to a method of preparing the emulsion based contrast agent. BACKGROUND OF INVENTION

[0002] A computed tomography scan (CT scan) is a medical imaging technique used to obtain detailed internal images of the body. CT scan uses a combination of X-rays and computer technology to produce images inside the body. CT scanning technology has revolutionized diagnostic imaging technology and contributed significantly to deepen our understanding of various diseases.

[0003] Contrast agents are substances used for diagnostic imaging procedures for enhancing the visibility of vascular structures and organs and have various applications in diagnostic imaging. The contrast agents currently in use in CT imaging are based on heavy metals. These substances can be administered to the patient orally, rectally, or intravenously. Common contrast material substances include iodine, barium, and gadolinium.

[0004] Iodine-based contrast agents available in the market are imported and water-soluble (Iohexol, Iopamidol, Iodixanol, Iopromide, Ioxilan, etc). The high contrast media concentrations required for CT place it at a disadvantage as compared to other imaging techniques such as MRI (mmol conc. range), nuclear imaging (µmol conc. range), and optical imaging (nmol conc. range). Some of the other drawbacks include low residence time for clear detection of tumour and incompatibility to be used in renal insufficiency patients.

[0005] Oil based contrast agents available in the market are primarily made of poppyseed oil, such as lipiodol. These are liposomes co-loaded with water-soluble iopamidol and water- insoluble iodinated poppy-seed oil. The oil-based contrast agent cannot be given intravenouslysince the oil particle may cause blocks in the arteries which may lead to myocardial infraction. Hence the oil-based contrast dyes are generally injected into the hepatic artery.

[0006] WO2015188040A2 relates to a composition for use in imaging. The compound or composition of the invention, which can be applied to any surface, can be used for visualizing surfaces, biological tissue, structures, and organs that would not necessarily be visible on an image produced by an imaging procedure.

[0007] US5512268A relates to polymeric shells for medical imaging prepared from synthetic polymers. The composition disclosed is useful as contrast agents for magnetic resonance imaging (MRI), ultrasonography, and X-ray computed tomography.

[0008] The polyunsaturated fatty acids and the form of iodine used in these products cause deleterious effects in kidneys and other vital organs such as lipid accumulation and free radical generation in organs. Moreover, the contrast agents are imported, and therefore, highly priced and unaffordable for common man. Further, contrast agents available cannot be used in renal insufficiency patients as it is toxic to the kidneys.

[0009] There are no emulsion based contrasts dyes available in the market at present, though studies related to Perfluorocarbon Emulsion Contrast Agents for CT, MRI, X-RAY are mentioned in research papers (Holman R et al., 2022). Oil-based contrast agents available in the market are mainly made of popyseed oil (Eg:- Lipiodol). However, the polyunsaturated fatty acids and the form of iodine used in these products cause deleterious effects on the kidney and other vital organs as well as lipid accumulation and free radical generation in organs. Moreover, the product is highly priced and is unaffordable for the common man. The available contrast agents in the market are water-based for cardiac purpose and the one oil-based are used as a biomarker for hepatic tumour detection, Trans Arterial Chemo Embolization (TACE), Glue dilution which is imported and highly-priced.

[0010] As a consequence, search for an optimal CT contrast agent with maximum imaging capabilities, minimal dose requirements, and reduced toxicity is an ongoing task.

[0011] The present invention discloses a composition of a contrast agent, which has reduced toxicity and is compatible to be used for renal insufficiency patients. SUMMARY OF INVENTION

[0012] Contrast agents are mainly used for diagnostic imaging procedures for enhancing the visibility of vascular structures and organs and have various applications in diagnostic imaging. Present disclosure relates to a lipid nano-emulsion-based contrast agent which is a novel non- ionic computed tomography (CT) contrast agent.

[0013] The composition of the contrast agent of the present invention comprises medium chain triglyceride, alpha-tocopherol, 2, 3, 5-triiodobenzoic acid and polyethylene glycol. Animal studies showed that the composition provides good imageability which is comparable to the existing product in the market. It can be used for multiple applications such as CT imaging for diagnosis of atherosclerotic lesions and tumour detection even in renal insufficiency patients.

[0014] Thus, it is one object of the present invention to provide a least toxic contrast dye for use in CT imaging.

[0015] It is another object of the invention to provide a method of preparing the least toxic contrast dye for use in CT imaging.

[0016] It is a further objective of the invention to provide a cost-effective contrast agent which does not compromise with quality.

[0017] It is yet another objective of the invention to provide a contrast agent for different imaging techniques such as CT imaging, for diagnosis of atherosclerotic lesions, hepatic tumor detection especially in renal insufficiency patients.

[0018] The above objectives of the invention are achieved through the below embodiments of the present invention.

[0019] In one embodiment A, a contrast agent for computed tomography is provided, comprising, 30 - 40% of 2, 3, 5-triiodobenzoic acid, 20 -25% of α-tocopherol, 10 - 20 % coconut oil, and 3 -10% polyethylene glycol.

[0020] Embodiment B relates to the contrast agent of previous embodiment, wherein the contrast agent is in the form of nano emulsion particles.

[0021] Embodiment C relates to the contrast agent of previous embodiment, wherein the nano- emulsion particles have a size ranging from 20 - 200nm.

[0022] Embodiment D relates to the contrast agent of the previous embodiment, wherein osmolality of the contrast agent ranges from 350-600 mOsmol.

[0023] Embodiment E relates a method of preparing a contrast agent as recited in previous embodiments, comprising steps of, preparation of α-tocopheryl 2,3,5- triiodobenzoate, preparation of an emulsion, incorporation of α-tocopheryl 2,3,5- triiodobenzoate with emulsion; wherein the step of preparation of α-tocopheryl 2,3,5- triiodobenzoate comprises, a) adding about 20 - 25 g of DL α-tocopheryl preferably 24.5g in to about 1600 - 2000ml of dicholoromethane, b) to the mixture of step a), add sequentially, 30-40g of 2,3,5-triiodobenzoic acid, 1-1.5g of 4-dimethylaminopyridine preferably 1.25g and 14 – 18g of N,N’-dicyclohexylcarbodiimide, preferably 16.1g at an atmospheric temperature of about 25-30˚C, c) the reaction mixture from step b) was stirred overnight at an atmospheric temperature of about 25-30˚C and the dicyclohexylurea and other precipitates were removed by filtration technique, d) organic phase which contains iodine from step c) was washed twice with saturated aqueous NaHCO3, once with saturated NaCl solution and dried with anhydrous Na2SO4, e) the solvent from step d) was removed in vacuum filtration using 0.4 to 0.5 micron filter paper, preferably 0.45 micron and the residue which containiodine was then purified by gradient elution, to yield α-tocopheryl 2,3,5- triiodobenzoate wherein the step of preparation of an emulsion comprises, i) adding about 0.5 to 1.0 g egg yolk lecithin preferably 0.6g to about 25- 35ml of deionised, pyrogen free water, wherein the water is pre heated to about 55-60˚C and maintained at about 60˚C and dissolved to obtain lecithin solution; ii) about 0.5 to 1.0g of sodium oleate preferably 0.75g is added to about 30 ml of deionised pyrogen free water, wherein the water is preheated to 55-60˚C and dissolved to obtain sodium oleate solution; iii) about 1.5 to 2.5 g of glycerol preferably 2.2g is added to sodium oleate solution obtained at step ii). iv) add lecithin solution of step i) to sodium oleate - glycerol solution of step iii) and mix and maintain at 55-60˚C to obtain an emulsifying solution, v) add about 10 to 20 g of coconut oil preferably 15g with addition of 22- 25 mg alpha tocopherol (Vitamin E); vi) the oil mixture of step v) is added drop by drop to emulsifying solution of step iv) with intermittent stirring and maintaining the temperature at 55-60˚C to obtain an emulsion; wherein the step of incorporation of α-tocopheryl 2,3,5- triiodobenzoate with emulsion comprises, A) to the solution of step v) above, add α-tocopheryl 2, 3, 5- triiodobenzoate of step e), B) the solution of step A) is sonicated at 55-60˚C temperature to obtain a fine emulsion based contrast agent, C) sterilize the contrast agent of step B) at 12-16 lbs pressure for 15-30 minutes, D) store the contrast agent obtained at step C) at temperature range of 8-25˚C. Embodiment F relates to the method as recited in previous embodiment, wherein gradient elution is carried out on silica gel using cyclohexane and ethyl acetate as an eluent in the ratio of 5:1.The above embodiments will be discussed in detail through figures and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] The foregoing summary, as well as the following detailed description of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of assisting in the explanation of the invention, they are shown in the drawings, embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown therein. In the drawings:

[0025] Fig 1 (A-C) shows sterility of the contrast agent in different media such as A) nutrient agar, B) soya bean casein and C) potato dextrose agar;

[0026] Fig 2 shows a graph of particle size of emulsion-based contrast dye using DLS (Dynamic Light Scattering);

[0027] Fig 3 (A-D) shows images of particle size of emulsion-based contrast dye from different areas of the same solution and analysed using TEM (Transmission Electron Microscope) A) Particles size between 52 nm to 80 nm, B) Particle size between 100 to 150nm, C) Particle size between 100nm to 150nm D) Particle size between 150 to 300nm;

[0028] Fig 4 (A-E) illustrates images of stability test of the contrast agent after orbital shaking; The orbital shaking stability of the nano-emulsion was examined through size distribution in function with incubation time for 1hr (B), 6hrs (C), 12hrs (D), 24hrs (E) and (A) representing contrast agent before shaking;

[0029] Fig 5 (A-D) illustrates images to show thermal stability of the contrast agent at room temperature; The test was repeated for different intervals of time (ie, 30 mins (B), 8hrs(C), 24hrs (D), and (A) representing sample before testing thermal stability at 40°C;

[0030] Fig 6 (A-D) illustrates stability of the contrast agent after centrifugation; (A) represents the sample before centrifugation, (B) after 30mins of centrifugation, and the cycle was repeated two more times (C and D);

[0031] Fig 7 shows imaging of the contrast agent in accordance with the invention;

[0032] Fig 8 shows imaging of the comparator product; (Iohexol, 350 mgI / ml);

[0033] Fig 9 (A-D) illustrates graphical representation of studies on acute toxicity of the contrast dye in liver and renal functions after two weeks; A) urea and uric acid, B) creatinine, C) ALP,SGOT,SGPT, D) tot. protein, albumin, globulin and s. bilirubin.

[0034] Fig 10 (A-E) illustrates graphical representation of studies on chronic toxicity of the contrast dye in liver and renal functions after two weeks; A) chloride and sodium, B) urea and calcium, C) creatinine, D) SGOT, SGPT and ALP, E) tot. protein, albumin and tot. billirubin.

[0035] Fig 11 shows micro-CT of liver of NOD SCID mice (11a. contrast agent in accordance with invention and 11b. comparator). DETAILED DESCRIPTION OF INVENTION

[0036] The present invention will now be described more fully herein after. For the purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified systems or embodiments that may of course, vary. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

[0037] As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0038] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0039] When the term “about” is used in describing a value or an endpoint of a range, the disclosure should be understood to include both the specific value and endpoint referred to.

[0040] As used herein the terms “comprises”, “comprising”, “includes”, “including”, “containing”, “characterized by”, “having” or any other variation thereof, are intended to cover a non-exclusive inclusion.

[0041] The term ‘room temperature’ or ‘atmospheric temperature’ referred herein is a temperature of 25-30˚C.

[0042] Contrast agents are mainly used for diagnostic imaging procedures for enhancing the visibility of vascular structures and organs and have various applications. Most of the iodine- based contrast agents available in the market are water soluble (for angiography) and the main limitation of these contrast agents is the low efficiency to detect tumours. Oil-based contrast agent available in the market is primarily made of popyseed oil (for Trans Arterial Chemo Embolization and glue dilution), which is not preferred for renal insufficiency patients as it can cause endothelial dysfunction. Moreover, the product is highly priced and is unaffordable for common man.

[0043] Inventors of the invention have successfully formulated an indigenous, non-toxic, safe and cost effective nano lipid-emulsion based contrast agent for preclinical X-ray imaging. The specific inventive steps are by formulating a non-toxic lipophilic molecule naturally present in the body α tocopheryl (Vit E) to graft a high concentration of X-ray contrasting material ie, 2, 3, 5-triodobenzoic acid using simplest chemical reaction. A nano lipid-emulsion based contrast dye was formulated using this alpha tocopheryl 2, 3, 5-triodobenzoic acid compound incorporating hairy Polyethylene glycol (PEG) layer to achieve stealth properties to avoid opsonization and enhance permeability and retention (EPR) effect.

[0044] The composition of the contrast agent as per the present invention comprises 2, 3, 5- triiodobenzoic acid, α-tocopherol, coconut oil and polyethylene glycol.2, 3, 5-triiodobenzoic acid is the iodine source in the composition. The composition comprises 30 - 40% of 2, 3, 5- triiodobenzoic acid. The iodine helps to increase the contrast in tissues by photoelectric absorption. The iodine compound used in this invention is fat soluble and is least toxic compared to the commercially available water-soluble contrast agents.

[0045] α-tocopherol is used in the composition as an antioxidant. The α-tocopherol or vitamin E combines with coconut oil and iodine to form iodinated α-tocopherol nano-emulsions. The composition comprises 20 -25% of α-tocopherol. The presence of α-tocopherol or vitamin E, which is an antioxidant with a nephroprotective effect.

[0046] Coconut oil is used in the composition as a source of medium chain fatty acid. About 60% of total fatty acid composition of coconut oil are medium-chain fatty acids (MCFA) with a chain length of 6 to 12 carbon atoms. The contrast agent composition of the present invention comprises 10 - 20 % coconut oil. The oil interacts with other components in the composition to form an emulsion.

[0047] The contrast agent of the present invention also includes polyethylene glycol (PEG). PEG comprises 3 -10% of the composition. The contrast agent is a lipid nano-emulsion which is formulated using alpha tocopheryl and 2, 3, 5-triodobenzoic acid compound, surrounded by hairy polyethylene glycol (PEG) layer to achieve stealth properties to avoid opsonisation and to enhance the permeability and retention of the contrast agent.

[0048] It is another objective of the invention to provide a method of preparing a contrast agent for use in computed tomography. The contrast agent in accordance with the present invention is prepared in three main steps of stages. The first step is to prepare the contrast dye by preparing a mixture of α-tocopheryl 2, 3, 5- tri-iodobenzoate. The second step is to prepare an emulsifying agent and the third step is incorporating the mixture of α-tocopheryl 2, 3, 5- triiodobenzoate from first step and the emulsifying agent from the second step together to form the contrast agent as per the present invention.

[0049] The first step in the preparation of the contrast agent is the preparation of α-tocopheryl 2, 3, 5- triiodobenzoate. To about 1600-2000ml of dicholoromethane, about 20 - 25g of DL α- tocopheryl, preferably 24.5g added and mixed well. To this mixture, about 30-40g of 2, 3, 5- triiodobenzoic acid, about 1 - 1.5g of 4-dimethylaminopyridine, preferably 1.25g and about 14 -18g of N,N’-dicyclohexylcarbodiimide, preferably 16.1g is added sequentially at room temperature. This reaction mixture was stirred overnight at an atmospheric temperature of about 25-30˚C and the dicyclohexylurea and other precipitates were removed by vacuum filtration, one of the best time-saving filtration method using filter paper of 0.4 - 0.5 micron size, preferably 0.45 micron size.

[0050] The organic phase which contains iodine is collected after filtration and is washed twice with saturated aqueous NaHCO3, once with saturated NaCl solution and dried with anhydrous Na2SO4. The resultant solvent from this step was removed under vacuum condition using a rotary evaporatorat 39 - 40 ˚C resulting in the formation of a yellow viscous oil, which was then purified by gradient elution, to yield α-tocopheryl 2,3,5- triiodobenzoate, which is a light, yellowish viscous oil with a high iodine content.

[0051] The gradient elution in the above discussed step is carried out on silica gel using cyclohexane and ethyl acetate as an eluent in the ratio of 5:1, using chromatography technique.

[0052] The second step in the process of preparing contrast agent as per the present invention is preparation of emulsifying agent. De-ionised, pyrogen free water is heated to about 55-60 ˚C temperature. To about 25-35 ml of de-ionised, pyrogen free water of temperature of about 55- 60 ˚C, about 0.5 - 1.0 g preferably 0.6 g egg yolk lecithin is added. The temperature of the water is maintained, and the lecithin is dissolved by stirring, to obtain lecithin solution.

[0053] To about 25-35 ml of deionised, pyrogen free water, maintained at a temperature of 55- 60˚C, about 0.5-1.0g of sodium oleate preferably 0.75g is added. Sodium oleate is dissolved in the water which is maintained at a temperature of 55-60˚C, by stirring the solution, to obtain sodium oleate solution. To the sodium oleate solution, about 1.5 - 2.5gof glycerol preferably 2.2g is added.

[0054] The lecithin solution and sodium oleate - glycerol solution is mixed and maintained at 55 - 60˚C to obtain an emulsion. To this emulsion, add about 10 – 20g preferably 15g of coconutoil with an addition of 22-25 mg alpha tocopherol (vitamin E). The oil mixture from the previous step is added drop by drop to the emulsifying solution, with intermittent stirring along with maintaining the temperature at 55-60˚C to obtain the emulsion.

[0055] The third step of preparation of the contrast agent includes incorporation of α- tocopheryl 2, 3, 5- tri-iodobenzoate with emulsion. To the emulsion solution comprising coconut oil and vitamin E obtained in second step, α-tocopheryl 2,3,5- triiodobenzoate obtained from first step is incorporated. The incorporated mixture is sonicated at 55-60˚C temperature to obtain a fine emulsion particles of nano size range. The emulsion obtained has a size of 20 - 200nm.

[0056] The nano sized emulsion particle which is the contrast agent is sterilized using autoclave at 12-16 lbs pressure for 15-30 minutes. The sterilized contrast agent is stored at temperature ranging from 8-25˚C, till use. EXAMPLES

[0057] Example 1: Sterility test

[0058] The sterility test of the contrast agent was tested by inoculating the same in separate petri dishes containing nutrient agar, soyabean casein and potato dextrose agar medium (Figure 1). The sample is spread on different media plates and incubated for 24hrs in a microbial incubator. Nutrient agar medium is used for checking the presence of bacteria, soyabean casein medium for checking the presence of aerobes and fungi and potato dextrose agar medium for checking the presence of fungal growth. The absence of growth of organisms in the plates showed that the sample is sterile.

[0059] Example 2: Stability test

[0060] Referring to Fig 4(A-E) the stability of the contrast dye was analyzed using temperature (thermal stability), and results showed that the proposed product is stable. Thermal stability (Fig 5) was performed to confirm the storage temperature of the contrast agent. The test was repeated for different intervals of time (ie, 30 mins (B), 8hrs(C), 24hrs (D), and (A) representing sample before testing thermal stability at 40°C. Stirring at 3500 rpm for 20 minutes could be said equivalent to gravity for ±1 year.

[0061] Example 3: Emulsion stability – centrifugation test

[0062] Referring to Fig5(A-D) the stability of the contrast dye was analysed using centrifugation. After three centrifugation cycles the emulsion showed good stability and results showed that the proposed product is stable. In Fig 6, (A) represents the sample before centrifugation, (B) after 30mins of centrifugation, and the cycle was repeated two more times (C and D). No separation of particles was observed until the last cycle, which indicated that the sample is stable.

[0063] Example 4: Emulsion stability –Orbital shaking test

[0064] Referring to Fig 4 (A-E) the stability of the contrast dye was analysed using orbital shaking and results showed that the proposed product is stable. The orbital shaking stability (Fig 4) of the nano-emulsion was examined through size distribution in function with incubation time for1hr (B), 6hrs (C), 12hrs (D), 24hrs (E) and (A) representing contrast agent before shaking. The samples were constantly homogenized to avoid sedimentation of large aggregates. Before analysis, the presence of aggregates was examined visually.

[0065] Example 5: Evaluation of particle size of nano emulsion contrast agent using DLS

[0066] The samples, 1 mL, were collected from the TPN bags, under laminar flow hood, in a clean room, with sterile syringes and needles and appropriately diluted with filtered WFI (0.2 μM) 1:500 to obtain the Kcps (a measure of the sample concentration, obtained by the equipment) from 250 to 350. Each analysis was performed in triplicate, using TPN prepared on different days. DLS measurements were carried out using a Zetasizer 3000Hs (Malvern Instruments, Worcestershire, United Kingdom), which uses a helium-neon laser light and an integrated analysis software. The temperature was adjusted to 25°C and the scattering angle was set to 90°, in the DLS equipment, before measurements were taken. The chosen data analysis method was the monomodal or cumulant analysis

[0025] . The data were expressed as z- average and polydispersity. The samples, 1 mL, were collected from the TPN bags, under laminar flow hood, in a clean room, with sterile syringes and needles and appropriately diluted with filtered WFI (0.2 μM) 1:500 to obtain the Kcps (a measure of the sample concentration, obtained by the equipment) from 250 to 350. Each analysis was performed in triplicate, using TPN prepared on different days. DLS measurements were carried out using a Zetasizer 3000Hs (Malvern Instruments, Worcestershire, United Kingdom), which uses a helium-neon laser light and an integrated analysis software. The temperature was adjusted to 25°Cand the scatteringangle was set to 90°, in the DLS equipment, before measurements were taken. The chosen data analysis method was the monomodal or cumulant analysis

[0025] . The data were expressed as z-average and polydispersity

[0067] 1 mL sample was taken, under laminar flow hood, in a clean room, with sterile syringes and needles and appropriately diluted with filtered WFI (0.2 μM) 1:500 to obtain the Kcps (a measure of the sample concentration, obtained by the equipment) from 250 to 350. DLS measurements were carried out using a Zetasizer 3000Hs (Malvern Instruments, Worcestershire, United Kingdom), which uses a helium-neon laser light and an integrated analysis software. The temperature was adjusted to 25°C and the scattering angle was set to 90°, in the DLS equipment, before measurements were taken. The chosen data analysis method was the monomodal or cumulant analysis. The data was expressed as z-average and polydispersity.

[0068] Example 6: Evaluation of particle size of nano emulsion contrast dye using TEM

[0069] Small quantity of specimens is optimally placed rapidly into glutaraldehyde and fixed for at least 1 hr at room temperature (23 °C) and then post-fixed in osmium tetroxide. Fixed cells are embedded in agar and processed based on the tissue type. The specimens are dehydrated in graded concentrations of ethanol and propylene oxide, and embedded in Spurr's plastic. Semi-thin sections are cut from blocks with a glass knife and the blocks are selected for thinning. Thin sections cut with diamond knives are placed on copper grids, impregnated with uranyl acetate and lead citrate, and examined. The image can be studied directly by the operator or photographed with a camera. A scale bar on a TEM image is essential to calculate the actual size of structures in the image.

[0070] Example 7: Osmolality test

[0071] Osmolality was assessed by vapor pressure osmometry (Vapro 5600, Wescor) with the instrument calibrated with vendor-supplied standards prior to measurement of triplicate samples, which were pipetted to the sample holder as undiluted 10-μl aliquots. Osmolality of proposed product found to be in between 396 and 575 mOsmol / kg. Osmolality values higher than 600 mOsmol / kg has been associated to hypertonicity-induced pain. Table 1 shows osmolality of the contrast dye (A5 and B5 - sample code).

[0072] Table 1 shows osmolality of the contrast dyeSl. No Sample ID Test Instrument Result Used 1 A5 Osmolality Osmomat 396 mOsmol / kg 3000 - 2 B5 575 mOsmol / kg Gonotec

[0073] Example 8: X-ray imaging of the contrast agent as per the invention and comparator product (Iohexol, 350 mgI / ml).

[0074] The animals were manually restrained and anesthetized using Xylazine at the rate of 5mg / kg body weight and Ketamine at the rate of 80mg / kg body weight intraperitoneally by mixing both the drugs in an insulin syringe. After the animals were completely under anesthesia, pedal pinch and tail flinch reflexes were checked and ensured to be abolished. Under sterile precautions, a surgical laparotomy was performed and the intestines were exteriorised and kept in wrapped in a sterile gauze cloth moistened with warm sterile saline. After bluntly dissecting the fat pads, abdominal aorta was dissected free from attachments and using a right-angled forceps, 2 ties (3.0, Mersilk) were passed beneath the abdominal aorta, making the vessel ready to be cannulated. Sterile heparinised normal saline was prepared and was kept ready to be injected to prevent clots during angiographic radiographic procedure.

[0075] Example 9: Micro CT imaging of liver of NOD SCID mice.

[0076] Hepatic tumor created in NOD SCID mice by using GFP tagged HepG2 cell line. After 4 months of study animals were injected with Emuloid and Comparator product. After 3 hrs of injection, animals were sacrificed and liver collected for micro CT imaging. The liver tissue which suspended in saline was blot dried. The micro CT of the specimen was observed using MILabs type U-CT instrument.

[0077] Example 10: Histopathological analysis of contrast agent administrated liver of NOD-SCID mice.

[0078] Hepatic tumor created in NOD SCID mice by using GFP tagged HepG2 cell line. After 4 months of study animals were injected with Emuloid and Comparator product. After 3 hrs of injection, animals were sacrificed, and liver collected for histology to check whether tumour is created or not. The fixed specimens of the liver was processed overnight fordehydration, clearing, and impregnation using an automatic tissue processor (Sakura, Japan). The specimens were embedded in paraffin blocks using an embedding station (Sakura, Japan) and serial sections of 4 µm thickness were cut using a microtome (ModelRM2245, Leica Biosystems, Wetzlar, Germany). We used an autostainer (Model 5020, Leica Biosystems, Wetzlar, Germany) for Hematoxylin and Eosin staining of the sections. The mounted specimens were observed and were scored under light microscopy. Table 2 shows Histopathological analysis of contrast agent administrated liver of NOD-SCID mice.

[0079] Table 2 shows Histopathological analysis of contrast agent administrated liver of NOD-SCID mice. Samples Observation Inferences Control Widespread vascular congestion, Early stage centrilobular degeneration, a few dysplastic of tumor foci with multinucleated hyperchromatic identified cells with cytologic atypia, Emulsion- Widespread hepatic degeneration, vascular Early stage based congestion, sub-capsular infiltration of of tumor contrast hyperchromatic cells with nuclear identified dye pleomorphism invading into the parenchyma Comparator Centrilobular degeneration with Early stage widespread vascular congestion, fatty of tumor changes, multifocal contoured identified hyperchromatic lesions, blood vessels invaded with hyper chromatic hepatic cells and mononuclear cells

[0080] Example 11: Evaluation of biochemical studies of emulsion based contrast dye in rats - chronic toxicity (8 weeks)

[0081] After 2 and 8 weeks of study, did not observe any toxicity in renal function test and liver function test functions. From these results we can conclude that, the proposed product can be given even to renal insufficiency patients.

[0082] Referring to Fig 9 (A-D), the acute toxicity (2 weeks) of the contrast dye in liver function (T. Billirubin, SGOT, SGPT, ALP, Total protein, albumin and globulin) and renal function (urea, creatinine and uric acid) in comparison with the control showing no toxicity.

[0083] Referring to Fig 10 (A-E), the chronic toxicity (8 weeks) of the contrast dye in liver function and renal function in comparison with the control showed no toxicity. After 8 weeks animal blood was collected, serum separated and subjected to liver function tests (T.Billirubin, Dir.Billirubin, SGOT, SGPT, ALP, Total protein, albumin and globulin) renal functions (urea, creatinine and calcium) and electrolytes (Chloride, Sodium, Potassium and Phosphorous).

[0084] Even after 8 weeks, the serum creatinine level (Fig 10 C) was found to be normal as compared to control. This indicated that the proposed product did not show any Contrast Induced Nephropathy (CIN).

[0085] Patients may experience renal function issues, such as elevated creatinine levels and the development of contrast-induced nephropathy (CIN), following CT scans. Therefore, the proposed product can safely be utilized for CT imaging even in patients with renal insufficiency.

[0086] The contrast agent of the present invention shows outstanding contrasting properties, biocompatibility and low toxicity, long residence time and high opacification. The form of iodine used in the composition is fat-soluble and very less toxic compared to the commercially available water-soluble contrast agent which uses a more toxic form. Preclinical studies in animal studies showed that the contrast agent as per the invention has good imageability which is comparable to the existing product in the market. Further, the contrast agent of the present invention can be used for multiple applications such as CT angiography of atherosclerotic lesions / occlusion and hepatic tumor detection even in renal insufficiency patients. The contrast agent in accordance with the invention is sterile and stable for more than six months. It shows low osmolarity. Characterization study shows an optimum range of particle size and the pharmacodynamics study showed no deleterious effects. Thus, it is safe to use, cost-effective and is developed indigenously compared to the existing imported product in the market.

[0087] While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as claimed.

Claims

AMENDED CLAIMS received by the International Bureau on 12 April 2025 (12.04.2025)WE CLAIM:

1. A contrast agent for computed tomography comprising,30 - 40% of 2, 3, 5 -triiodobenzoic acid,20 -25% of a-tocopherol,10 - 20 % coconut oil, and3 -10% polyethylene glycol wherein the contrast agent is in the form of nano emulsion particles having a size ranging from 20 - 200nm; the emulsion comprises emulsifying agents egg yolk lecithin, sodium oleate and glycerol.

2. The contrast agent as claimed in claims 1, wherein osmolality of the contrast agent ranges from 300 - 600 mOsmol.

3. A method of preparing a contrast agent as claimed in claims 1- 2, comprising steps of, preparation of a-tocopheryl 2,3,5- triiodobenzoate, preparation of an emulsion, incorporation of a-tocopheryl 2,3,5- triiodobenzoate with emulsion; wherein the step of preparation of a-tocopheryl 2,3,5- triiodobenzoate comprises, a) adding about 20 - 25 g of DL a-tocopheryl preferably 24.5g in to about 1600 - 2000ml of dicholoromethane, b) to the mixture of step a), add sequentially, 30-40g of 2,3,5-triiodobenzoic acid, 1-1.5g of 4-dimethylaminopyridine preferably 1.25gand 14 - 18g of N,N’ -dicyclohexylcarbodiimide, preferably 16.1g at an atmospheric temperature of about 25 -30 °C, c) the reaction mixture from step b) was stirred overnight at an atmospheric temperature of about 25-30°C and the dicyclohexylurea and other precipitates were removed by fdtration technique, d) organic phase which contains iodine from step c) was washed twice with saturated aqueous NaHCOv once with saturated NaCl solution and dried with anhydrous Na2SC>4, e) the solvent from step d) was removed in vacuum fdtration using 0.4 to 0.5 micron fdter paper, preferably 0.45 micron and the residue which contain iodine was then purified by gradient elution, to yield a-tocopheryl 2,3,5- triiodobenzoate; wherein the step of preparation of an emulsion comprises,i) adding about 0.5 to 1.0 g egg yolk lecithin preferably 0.6g to about 25- 35ml of deionised, pyrogen free water, wherein the water is pre heated to about 55-60°C and maintained at about 60°C and dissolved to obtain lecithin solution; ii) about 0.5 to 1.0g of sodium oleate preferably 0.75g is added to about 30 ml of deionised pyrogen free water, wherein the water is preheated to 55-60°C and dissolved to obtain sodium oleate solution; iii) about 1.5 to 2.5 g of glycerol preferably 2.2g is added to sodium oleate solution obtained at step ii). iv) add lecithin solution of step i) to sodium oleate - glycerol solution of step iii) and mix and maintain at 55-60°C to obtain an emulsifying solution, v) add about 10 to 20 g of coconut oil preferably 15g with addition of 22- 25 mg alpha tocopherol (Vitamin E); vi) the oil mixture of step v) is added drop by drop to emulsifying solution of step iv) with intermittent stirring and maintaining the temperature at 55-60°C to obtain an emulsion; wherein the step of incorporation of a-tocopheryl 2,3,5- triiodobenzoate with emulsion comprises,A) to the solution of step v) above, add a-tocopheryl 2, 3, 5- triiodobenzoate of step e),B) the solution of step A) is sonicated at 55-60°C temperature to obtain a fine emulsion based contrast agent,C) sterilize the contrast agent of step B) at 12-16 lbs pressure for 15-30 minutes,D) store the contrast agent obtained at step C) at temperature range of 8-25 °C.

4. The method as claimed in claim 3, wherein gradient elution is carried out on silica gel using cyclohexane and ethyl acetate as an eluent in the ratio of 5 : 1.STATEMENT UNDER ARTICLE 19 (1)We refer to the above-captioned PCT application.Further to the International Search Report and the Written Opinion of the International Searching Authority, issued and mailed on 14 February 2025, we hereby submit the amended claims under Article 19 and the basis for amendments.We hereby amend the claims to align the claims in line with the pending claims in India, and to overcome the objections stated in the International Search Report and the Written Opinion of the International Searching AuthorityA clean copy of the amended complete set of claims in replacement of the claims originally filed is enclosed. We are also submitting the amended claims with track changes for your ease of reference.Scope of claims 1, 2 and 3 have been merged to amended claim 1.Amended claim 1 also includes the feature that “the emulsion comprises emulsifying agents egg yolk lecithin, sodium oleate and glycerol”. The method claims already include this feature. Furthermore, support can also be found in paragraphs [0052] and [0053] of the PCT specification.No new matter has been added.A clean copy of the amended complete set of claims, replacing those originally filed, is enclosed herewith. For ease of reference, we are also submitting a marked-up version of the amended claims showing the changes made.We respectfully request that the enclosed documents be taken on record.

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